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LECTURE 4. MICROBIAL GROWTH

LECTURE 4. MICROBIAL GROWTH. 1. Microbial survival strategies 2. Microbial growth 3. Effects of the environment 4. Microbial cultures 5. Growth measurements 6. Antimicrobial agents 7. Antibiotics. 1. Microbial survival strategies. PROKARYOTES: MINIATURIZATION EUKARYOTES: COMPLEXITY.

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LECTURE 4. MICROBIAL GROWTH

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  1. LECTURE 4. MICROBIAL GROWTH 1. Microbial survival strategies 2. Microbial growth 3. Effects of the environment 4. Microbial cultures 5. Growth measurements 6. Antimicrobial agents 7. Antibiotics

  2. 1. Microbialsurvivalstrategies PROKARYOTES: MINIATURIZATION EUKARYOTES: COMPLEXITY Prokaryotes – r strategists: “the advantages of being small”  Surface/Volumen • Metabolic rates (osmotrophs) Rapid growth, short generation times Population-level adaptation Eukaryotes –K strategists: “the advantages of being complex” Phagocytosis Complexgenomes Rapid movement Independence fromtheenvironment Complex sensor and motor systems Individual-leveladaptation

  3. 2. MICROBIAL GROWTH GROWTH (IN MICROBIOLOGY)=INCREASE IN CELL NUMBER 2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS DNA replication and cell elongation Septum formation Completion of septum withformation of distinctwalls Cell separation Binary fission vs other

  4. 2. MICROBIAL GROWTH 2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS Partition of cell components: random (except DNA) PROTEINS Fts DNA SEGREGATION: DNA binds to the cytoplasmic membrane • Bidirectional replication (in one fork) • Several simultaneous forks

  5. 3. EFFECTS OF THE ENVIRONMENT 3.1. NUTRIENT CONCENTRATION At a verylownutrientconcentration, permeases cannotkeephighlevelsof nutrientsinsidethecell and growthratedecreases. However, highnutrientconcentrations can be toxicformanymicroorganisms 3.2. PREASURE Al sea level = 1 atm In the oceans up to 600 -1.100 atm • No barotolerant • Barotolerant • Barophile (or piezophile)

  6. 3. EFFECTS OF THE ENVIRONMENT 3.3. TEMPERATURE Enzymatic reactions occurring at maximum possible rate Enzymatic reactions occrring at increasingly rapid rates • Psychrophile ( 0 - 20ºC) • Mesophile (10 - 50ºC) • Termophile (50 - 70ºC) • Hyperthermophile (80 - 121ºC*) Membrane gelling; transport processes so slow that growth cannot occur Protein denaturation; collapse of the cytoplasmic membrane;thermal lysis Adaptations to high temperatures Adaptations to low temperatures Thermoresistent proteins (enzymes) Stable membranes ( saturated fatty acids) Archaea, special membranes (lipid monolayers) Proteins (enzymes) that function optimally in the cold Modified active transport processes Fluidity of membranes ( unsaturated fatty acids)

  7. 3. EFFECTS OF THE ENVIRONMENT 3.4. OXYGEN CONCENTRATION TOXIC OXYGEN SPECIES: Singlet oxygen (1O2) Superoxide (O2-) Hydrogen peroxide (H2O2) Hydroxyl radical (OH-) Enzymes that destroy them 1 2 3 4 5 Aerobic (1) Microaerophilic (aerobic) (4) Facultative (3) Aerotolerant anaerobe(5) (Strict) anaerobic (2)

  8. 3. EFFECTS OF THE ENVIRONMENT 3.5. pH

  9. 3. EFFECTS OF THE ENVIRONMENT 3.6. OSMOLARITY Adaptations to high salt • Counterbalance of external osmotic preasure by accumlation of: • Inorganic ions (K+) . Acidic proteins! Archaea and some extremely halophilic Bacteria. • Compatible organic solutes (either imported or synthesized): glycine betaine, proline, glycerol, etc.

  10. 3. EFFECTS OF THE ENVIRONMENT 3.7. RADIATIONS Photodynamic effect: light-mediated generation of singlet oxygen ( 1O2 ) Carotenoids: photoprotectant pigments transform 1O2 into non toxic species Radiotolerant microorganisms: Bacterial endospores Deinococcus radiodurans

  11. 3. EFFECTS OF THE ENVIRONMENT 3.8. EXTREMOPHILISM AND EXTREMOPHILES

  12. 4. MICROBIAL CULTURES CULTURE: a system used to allow the multiplication of a microbial population and reach a high microbial density Culture components: Nutrients (medium) Inoculum (absence of contamination) Culture types: Pure (or axenic) Mixed Preparation (According to the metabolic categories): C source E source Macronutrients (N, O, P, S, salts, vitamins, etc.) Micronutrients (normally, present as salt contaminants) H2O pH (buffers) WARNING! Auxotrophsvsprototrophs Sterilization Inoculation Types of culture media: Liquid / solid Defined, synthetic Complex Selective “Test”… Incubation

  13. 4. MICROBIAL CULTURES

  14. 4. MICROBIAL CULTURES 4.1. BATCH (DISCONTINUOUS) CULTURE Closedsystem(onlyenergy, and sometimesgases, are interchangedwiththeexternalenvironment; no cellsor disolved products). Growth curve with 4 phases.

  15. 4. MICROBIAL CULTURES 4.1. BATCH CULTURE Exponential growth • N = N02n • N= Number of cellsafter n generations • N0 = Number of cellsat the beginning • tg = Generation time • (time neededtodoublecellnumber) • = specificgrowthrate (time units-1) • (number of generations per time unit) • tg = ln 2 /  (hours)

  16. 4. MICROBIAL CULTURES 4.1. BATCH CULTURE

  17. 4. MICROBIAL CULTURES 4.1. BATCH CUTURE Escherichia coli Tg= 20 min After 48 hours 4000 X Earth weight µ depends on nutrient concentration LIMITING SUBSTRATE Net growth: final biomass – initial biomass (inoculum) Yield: unit of biomass produced per unit of nutrient consumed Y = (X – Xo) / S Y = yield X, Xo = cells/ ml S = nutrient concentration at to S µ = µmax Ks + S

  18. 4. MICROBIAL CULTURES 4.2. CONTINUOUS CULTURE (“steady state”) Cultures can be kept for long periods of time. Medium is added and culture removed, keepin V constant V entrance constant, [nutrient] constant, [cell] constant. V changes, µ changes and a new [cell] is reached

  19. 4. MICROBIAL CULTURES 4.2. CONTINUOUS CULTURE (“steady state”) Biomass (X) constant in time. dX/dt = 0 Vproduction (cells produced) = Vlosses (cells removed)

  20. 4. MICROBIAL CULTURES 4.3. CULTURES ON SOLID MEDIA

  21. 5. GROWTH MEASUREMENTS Only balanced growth(ordered increase of all cell components) can be measured properly… 5.1. BIOMASS Dry weight Absorbance (cell density)* 5.2. CELL COMPONENTS Nucleic acids, proteins, enzymatic activities 5.3. CELL NUMBRES Counting chamber Flow cytometer Total cells Plate counts Most probable number (MPN) Viable cells (culturable)*** ***VBNC: Viable but not culturable

  22. 5. GROWTH MEASUREMENTS 5.3. CELL NUMBERS How many microbes are present in a natural sample? “Who” are they? What do they do? (niche) How do they relate to each other and to other organisms? (competition, antagonisms, symbioses, etc.) Description of natural microbial communities Plate count (“viables”) SAMPLE Direct counts

  23. Problems encountered when counting “viables” (i.e. when culturing) Are theydead? Are they viable butnotculturable (VBNC)*? They do notgrowonstandard culture media *Important in public health

  24. 6. CONTROL OF MICROBIAL GROWTH ANTIMICROBIAL AGENTS: either (i) limit or inhibit microbial growth or (ii) destroy microorganisms IMPORTANT CONCEPTS • Sterilization: a processthatdestroysall living organisms and theirvirusesfromanobjectorhabitat. • Disinfection: partialeliminationorinhibition of microbes, normallypathogensDisinfectant: (chemical) agentsusedtodisinfect; usedoninanimateobjects. • Antisepsis:prevention of sepsis orinfection (antisepticagents are usedovertissuestopreventinfections, normallylesstoxicthandisinfectantagents). • Germicide:destroygerms (pathogens) and non-pathogens, butnotnecessarilyspores (bactericide, algaecide, fungicide, virocide...)

  25. 6. CONTROL OF MICROBIAL GROWTH ANTIMICROBIAL AGENTS

  26. 6. CONTROL OF MICROBIAL GROWTH 6.1. EFFECTS OF ANTIMICROBIAL AGENTS BACTERIOSTATIC BACTERICIDE BACTERIOLYTIC

  27. 6. CONTROL OF MICROBIAL GROWTH 6.2. FACTORS THAT AFFECT THE EFFICIENCY OF ANTIMICROBIAL AGENTS • Population size: the same fraction of the microbial population is destroyed in each time interval; a larger population needs more time to be completely eliminated than a smaller one. • Population composition: different microbes have different sensitivity to antimicrobial agents • Concentration and performance of the antimicrobial agent • Exposure time • Temperature • Local environment: pH, organic matter, biofilms, etc.

  28. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS MOIST HEAT Boiling in water for 10 minutes destroys vegetative cells and eukaryotic spores but NOT bacterial endospores Autoclave: temperatureshigherthan 100oC (pressure) withwatersaturatedsteam. Time: 10-15 minutes. Dependsonthesamplevolume.

  29. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS NO STERILIZATION PASTEURIZATION Foodtreatment (milk...) Old method: 63oC for 30 minutes. Fastpasteurization(HTST: high-temperature short-term): 72oC for 15 seconds. Sterilization at ultrahightemperature(UHT: ultra-hightemperature): 140-150oC for 1-3 seconds. DRY HEAT Oven at 160-170 oC from 2 to 3 hours Not suitable for thermosensitive materials Used for glass, oil and other materials Suele utilizarse para material de vidrio, aceite y otros materiales Clostridiumbotulinumendospores Moistheat: 5 min at 121 oC Dryheat: 2 hours at 160oC

  30. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS LOW TEMPERATURES Inhibit growth Used to preserve (not to sterilize or disinfect) FILTRATION

  31. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS RADIATION • UV (ceiling, biological safety hoods) • Ionizing radiation: very good sterilizing agent. • Pharmaceutical companies • Disposable clinical materials • Meat and other foods (spices) Comercial radiation of spices and seasonings, world data

  32. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS Germicides Disinfectant and antispetic Antibiotics SELECTIVE TOXICITY NO SELECTIVE TOXICITY • STERILIZING AGENTS • Ethylene oxide • Formaldehyde • Glutaraldehyde • H2O2 30% • DISINFECTANT AGENTS • Alcohols • Chlorinatedcompounds • Phenoliccompounds • H2O2 6% • ANTISEPTIC AGENTS • Mercury-containingcompounds • Iodine • H2O2 3% • Phenols • Alcohols • Halogenated compounds • Heavy metals • Aldehydes • Hydrogen peroxide • Surfactant agents • Ethiylene oxides • ANTIBIOTICS

  33. 6. CONTROL OF MICROBIAL GROWTH 6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS

  34. 6. CONTROL OF MICROBIAL GROWTH 6.5. MEASURING ANTIMICROBIAL ACTIVITY MINIMUM INHIBITORY CONCENTRATION

  35. 7. ANTIBIOTICS A chemical substance produced by a microorganism (fungi or bacteria) that kills or inhibits the growth of another microorganism. Normally, they have selective toxicity*: the ability of a compound to inhibit or kill pathogenic microorganisms without adversely affecting the host. Thus, they can be used as chemotherapeutical agents. Some antibiotics are semi-synthetic. “*The magic bullet” BACTERIOSTATIC BACTERICIDE BACTERIOLYTIC MAIN ANTIBIOTIC TARGETS Cell wall synthesis Protein synthesis Cell membrane integrity Nucleic acids synthesis Essential cofactors synthesis

  36. 7. ANTIBIOTICS ANTIBIOTIC MECHANISMS

  37. 7. ANTIBIOTICS

  38. 7. ANTIBIOTICS

  39. 7. ANTIBIOTICS 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS PENICILLINS (b-LACTAMIC) b-lactamic ring (degradedbyb-lactamasesorpenicillinases) Penicilina G Synthesized by the fungus Penicillium (Fleming, 1928) Bacteriolytic (destroy growing cells) They inhibit transpeptidation Bacteria can be resistant to penicillins if they synthesize penicillinases (b-lactamases) They can be combined with clavulanic acid

  40. 7. ANTIBIOTICS 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS PENICILLINS (b-LACTAMIC)

  41. 7. ANTIBIOTICS 7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS CEPHALOSPORINS Cephalosporium acreminium They inhibit transpeptidation

  42. 7. ANTIBIOTICS 7.2. PROTEIN SYNTHESIS INHIBITORS AMINOGLUCOSIDES TETRACICLINES Theeffectcan be reverted The effect cannot be reverted Examples: streptomycin, kanamycin, etc. MACROLIDES CHLORAMPHENICOL Erythromycin

  43. 7. ANTIBIOTICS 7.3. OTHER MECHANISMS Changes in the properties of the cell membrane: - Polymixin B Interference with nucleic acids synthesis: - Rifampicin (inhibits RNA polymerase) - Quinolones (inhibit DNA topoisomerases) Inhibit essential cofactors synthesis: - Sulfamides (inhibit folic acid synthesis) 7.4. BACTERIOCINS Agents produced by certain bacteria (or archaea) that inhibit or kill closely related species.

  44. 7. ANTIBIOTIC 7.5. ANTIFUNGAL AGENTS

  45. 7. ANTIBIOTICS 7.6. ANTIVIRAL

  46. 7. ANTIBIOTICS 7.7. ANTIBIOTIC RESISTANCE

  47. 7. ANTIBIOTICS 7.7. ANTIBIOTIC RESISTANCE RESISTANCE MECHANISMS • Theantibioticcannotreachits target • Theantibioticisdegradedormodified • Theantibiotic target ismodified Antibiotic resitance can be chromosomic (mutation) or plasmidic (transferable) Plasmids R Can we stop antibiotic resistance?

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